DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Lipid bilayer membrane permeability mechanism of the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 elucidated by molecular dynamics simulations.

Peptides are mid-size molecules (700-2000 g/mol) and have attracted particular interest as therapeutic modalities as they are superior in controlling protein-protein interactions, a process that is a typical drug target category, compared with small molecules (<500 g/mol). In 2020, we identified KS-58 (1333 g/mol) as a K-Ras(G12D)-inhibitory bicyclic peptide and suggested its cell membrane permeability. However, the membrane permeability mechanism had not been elucidated. In this study, we aim to clarify the mechanism by molecular dynamics (MD) simulations. Initially, we simulated the molecular conformations of KS-58 in water (a polar solvent) and in chloroform (a non-polar solvent). The identified stable conformations were significantly different in each solvent. KS-58 behaves as a chameleon-like molecule as it alters its polar surface area (PSA) depending on the solvent environment. It was also discovered that orientation of Asp's side chain is a critical energy barrier for KS-58 altering its conformation from hydrophilic to lipophilic. Taking these properties into consideration, we simulated its lipid bilayer membrane permeability. KS-58 shifted toward the inside of the lipid bilayer membrane with altering its conformations to lipophilic. When the simulation condition was set in deionized form of that carboxy group of Asp, KS-58 traveled deeper inside the cell membrane. PSA and the depth of the membrane penetration correlated. In vitro data suggested that cell membrane permeability of KS-58 is improved in weakly acidic conditions leading to partial deionization of the carboxy group. Our data provide an example of the molecular properties of mid-size peptides with membrane accessibility and propose an effective metadynamics approach to elucidate such molecular mechanisms by MD simulations.

Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3ß (GSK3ß), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3ß pathways, and mediates the G1/S transition to control cell proliferation.

Arnica montana L. extract containing 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin accelerates growth and differentiation of human subcutaneous preadipocytes and leads volumizing of skin.

OBJECTIVE: An important factor in the aging of the face is a reduction in the volume of adipose tissue. This reduction in adipose tissue contributes to decreased skin elasticity, which is also part of the aging process. Overall, these lead to wrinkle formation. Fat injection is a common means of addressing this issue and is used to reduce the effects of aging on the face and to increase the fullness of the lips and breasts. However, fat injection is an invasive surgical procedure. This study aimed to discover novel cosmetic ingredients that increase the volume of subcutaneous (pre)adipocytes to create the appearance of more youthful skin. METHODS: We focused on the number of subcutaneous preadipocytes and the accumulation of lipid droplets. To discover natural ingredients that increase both of these, extracts of 380 natural products were prepared and screened for their effects on both growth and differentiation (i.e., lipid droplet accumulation) of human subcutaneous preadipocytes. One extract was found to have the desired effects, and this was further studied to determine the active compounds. We then evaluated its efficacy in a human clinical study. RESULTS: We found that Arnica montana L. flower extract (AFE) accelerates both the growth and the differentiation of human subcutaneous preadipocytes. AFE was found to significantly increase the volume of adipocyte spheroids. The active compounds 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin were found to be responsible for the effects of AFE on preadipocytes. In a human clinical study, gels containing 1% AFE successfully enhanced the volume of the lips and face with reduction of wrinkles with no adverse reactions. CONCLUSION: This is the first report to demonstrate that AFE and the included compounds, 6-O-methacryloylhelenalin and 6-O-isobutyrylhelenalin, act on preadipocytes. AFE would be ideal for use in products that plump the face to reduce wrinkles and create a more youthful appearance.

OBJECTIF: Un facteur important du vieillissement du visage réside dans la réduction du volume du tissu adipeux. Cette réduction du tissu adipeux contribue à une diminution de l'élasticité de la peau qui fait également partie du processus de vieillissement. Globalement, ces facteurs induisent la formation des rides. L'injection de graisse est un moyen courant pour remédier à ce problème et sert à réduire les effets du vieillissement sur le visage et à augmenter la plénitude des lèvres et des seins. Cependant, l'injection de graisse est une intervention chirurgicale invasive. Cette étude visait à découvrir des ingrédients cosmétiques innovants qui augmentent le volume des (pré)adipocytes sous-cutanés pour créer l'apparence d'une peau plus jeune. MÉTHODES: Nous avons mis l'accent sur le nombre de préadipocytes sous-cutanés et sur l'accumulation de gouttelettes lipidiques. Pour découvrir des ingrédients naturels qui augmentent ces deux facteurs, des extraits de 380 produits naturels ont été préparés et analysés en vue de la détermination de leurs effets sur la croissance et la différenciation (c'est-à-dire l'accumulation de gouttelettes lipidiques) des préadipocytes humains sous-cutanés. Un extrait s'est avéré avoir les effets escomptés et il a fait l'objet d'études approfondies visant à déterminer les composés actifs. Nous avons ensuite évalué son efficacité dans une étude clinique chez l'homme. RÉSULTATS: Nous avons découvert que l'extrait de fleur de l'Arnica montana L. (AFE) accélère à la fois la croissance et la différenciation des préadipocytes humains sous-cutanés. L'AFE s'est avéré augmenter considérablement le volume des sphéroïdes des adipocytes. Les composés actifs 6-Ométhacryloyl-hélénaline et 6-O-isobutyryl-hélénaline se sont avérés responsables des effets de l'AFE sur les préadipocytes. Dans une étude clinique chez l'homme, des gels contenant 1 % d'AFE ont permis d'améliorer le volume des lèvres et du visage avec une réduction des rides sans effets indésirables. CONCLUSION: Il s'agit du premier rapport démontrant que l'AFE et les composés inclus, 6-O-méthacryloyl-hélénaline et 6-O-isobutyryl-hélénaline, agissent sur les préadipocytes. L'AFE serait idéal pour les produits qui repulpent le visage afin de réduire les rides et de donner un aspect rajeuni.

Juniper berry extract containing Anthricin and Yatein suppresses lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases spots in human skin.

OBJECTIVE: Skin brightness and spot have a significant impact on youthful and beautiful appearance. One important factor influencing skin brightness is the amount of internal reflected light from the skin. Observers recognize the total surface-reflected light and internal reflected light as skin brightness. The more internal reflected light from the skin, the more attractive and brighter the skin appears. This study aims to identify a new natural cosmetic ingredient that increases the skin's internal reflected light, decreases spot and provides a youthful and beautiful skin appearance. METHODS: Lipofuscin in epidermal keratinocytes, the aggregating complex of denatured proteins and peroxidized lipids, is one factor that decreases skin brightness and causes of spot. Aggregates block light transmission, and peroxidized lipids lead to skin yellowness, dullness and age spot. Lipofuscin is known to accumulate intracellularly with ageing. Rapid removal of intracellular denatured proteins prevents lipofuscin formation and accumulation in cells. We focused a proteasome system that efficiently removes intracellular denatured proteins. To identify natural ingredients that increase proteasome activity, we screened 380 extracts derived from natural products. The extract with the desired activity was fractionated and purified to identify active compounds that lead to proteasome activation. Finally, the efficacy of the proteasome-activating extract was evaluated in a human clinical study. RESULTS: We discovered that Juniperus communis fruits (Juniper berry) extract (JBE) increases proteasome activity and suppresses lipofuscin accumulation in human epidermal keratinocytes. We found Anthricin and Yatein, which belong to the lignan family, to be major active compounds responsible for the proteasome-activating effect of JBE. In a human clinical study, an emulsion containing 1% JBE was applied to half of the face twice daily for 4 weeks, resulting in increased internal reflected light, brightness improvement (L-value) and reduction in yellowness (b-value) and spot in the cheek area. CONCLUSION: This is the first report demonstrating that JBE containing Anthricin and Yatein decreases lipofuscin accumulation in human epidermal keratinocytes through proteasome activation, increases brightness and decreases surface spots in human skin. JBE would be an ideal natural cosmetic ingredient for creating a more youthful and beautiful skin appearance with greater brightness and less spot.

OBJECTIF: La luminosité et les taches de peau ont un impact significatif sur la jeunesse et la beauté de l'apparence. L'un des facteurs importants influençant la luminosité de la peau est la quantité de lumière interne réfléchie par la peau. Pour les observateurs, la luminosité de la peau correspond à la somme de la lumière réfléchie par la surface et de la lumière réfléchie par l'intérieur de la peau. Plus la quantité de lumière interne réfléchie par la peau est importante, plus la peau semble attrayante et lumineuse. Cette étude vise à identifier un nouvel ingrédient cosmétique naturel qui augmente la lumière interne réfléchie par la peau, diminue les taches et donne à la peau une apparence jeune et belle. MÉTHODES: La lipofuscine dans les kératinocytes de l'épiderme, le complexe agrégé de protéines dénaturées et de lipides peroxydés, est un facteur qui diminue l'éclat de la peau et qui est à l'origine des taches. Les agrégats bloquent la transmission de la lumière et les lipides peroxydés entraînent une coloration jaune de la peau, un aspect terne et des taches de vieillesse. On sait que la lipofuscine s'accumule au niveau intracellulaire avec le vieillissement. L'élimination rapide des protéines dénaturées intracellulaires empêche la formation et l'accumulation de lipofuscine dans les cellules. Nous avons mis l'accent sur un système de protéasome qui élimine efficacement les protéines dénaturées intracellulaires. Pour identifier les ingrédients naturels qui augmentent l'activité du protéasome, nous avons passé au crible 380 extraits dérivés de produits naturels. L'extrait présentant l'activité souhaitée a été fractionné et purifié afin d'identifier les composés actifs qui conduisent à l'activation du protéasome. Enfin, l'efficacité de l'extrait activant le protéasome a été évaluée dans une étude clinique humaine. RÉSULTATS: Nous avons découvert que l'extrait de Juniperus communis fruits (baie de genièvre) augmente l'activité du protéasome et supprime l'accumulation de lipofuscine dans les kératinocytes épidermiques humains. Nous avons découvert que l'anthricine et la yateine, qui appartiennent à la famille des lignanes, sont les principaux composés actifs responsables de l'effet activateur du protéasome de l'extrait de baies de genévrier. Dans une étude clinique humaine, une émulsion contenant 1 % de JBE a été appliquée sur la moitié du visage deux fois par jour pendant 4 semaines, ce qui a entraîné une augmentation de la lumière interne réfléchie, une amélioration de la luminosité (valeur L) et une réduction de la jaunisse (valeur b) et des taches dans la zone des joues. CONCLUSION: Il s'agit du premier rapport démontrant que l'EBJ contenant de l'anthricine et de la yateine diminue l'accumulation de lipofuscine dans les kératinocytes épidermiques humains par l'activation du protéasome, augmente la luminosité et diminue les taches superficielles de la peau humaine. Le JBE serait un ingrédient cosmétique naturel idéal pour créer une peau plus jeune et plus belle, plus lumineuse et moins tachetée.

AlphaFold version 2.0 elucidates the binding mechanism between VIPR2 and KS-133, and reveals an S-S bond (Cys25-Cys192) formation of functional significance for VIPR2.

The vasoactive intestinal peptide receptor 2 (VIPR2) has attracted attention as a drug target for the treatment of mental disorders, cancer, and immune diseases. In 2021, we identified the peptide KS-133 as a VIPR2-selective antagonist. In this study, we aimed to elucidate the binding mechanism between VIPR2 and KS-133. To this end, VIPR2/KS-133 and VIPR2/vasoactive intestinal peptide (VIP) complex models were constructed through AlphaFold version 2.0 and molecular dynamic simulations. Our models revealed that: (i) both KS-133 and VIP have helical structures, (ii) the interaction residues on VIPR2 for both peptides are similar, and (iii) the orientation of their helices upon their binding to VIPR2 are different by ∼45°. Interestingly, in the process of constructing the aforementioned models, an S-S bond formation between Cys25 and Cys192 of the human VIPR2 was identified. Although these two Cys residues are highly conserved among species (i.e., corresponding to Cys24 and Cys191 in the mouse), no previous reports regarding this S-S bond formation exist. In order to clarify the potential role of this S-S bond in the VIPR2 has functional consequences, a cell line expressing the mouse VIPR2(Cys24Ala, Cys191Ala) was generated. During the VIP stimulation of this cell line, the phosphorylation of AKT (a downstream signal marker of VIPR2) was found to be significantly attenuated, thereby suggesting that the S-S bond has a functional significance for VIPR2. Our study not only elucidates the VIPR2-binding mechanism of KS-133 for the first time, but also provides new insights into the structural biology of VIPR2.

Four-Types of IIT-Induced Group Integrity of Plecoglossus altivelis.

Integrated information theory (IIT) was initially proposed to describe human consciousness in terms of intrinsic-causal brain network structures. Particularly, IIT 3.0 targets the system's cause-effect structure from spatio-temporal grain and reveals the system's irreducibility. In a previous study, we tried to apply IIT 3.0 to an actual collective behaviour in Plecoglossus altivelis. We found that IIT 3.0 exhibits qualitative discontinuity between three and four schools of fish in terms of Φ value distributions. Other measures did not show similar characteristics. In this study, we followed up on our previous findings and introduced two new factors. First, we defined the global parameter settings to determine a different kind of group integrity. Second, we set several timescales (from Δ t = 5 / 120 to Δ t = 120 / 120 s). The results showed that we succeeded in classifying fish schools according to their group sizes and the degree of group integrity around the reaction time scale of the fish, despite the small group sizes. Compared with the short time scale, the interaction heterogeneity observed in the long time scale seems to diminish. Finally, we discuss one of the longstanding paradoxes in collective behaviour, known as the heap paradox, for which two tentative answers could be provided through our IIT 3.0 analysis.

Discovery of an Irreversible and Cell-Active BCL6 Inhibitor Selectively Targeting Cys53 Located at the Protein-Protein Interaction Interface.

B-cell lymphoma 6 (BCL6) is the most frequently involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 shows potent transcriptional repressor activity through interactions with its corepressors, such as BCL6 corepressor (BCOR). The inhibition of the protein-protein interaction (PPI) between BCL6 and its corepressors suppresses the growth of BCL6-dependent DLBCLs, thus making BCL6 an attractive drug target for lymphoma treatment. However, potent small-molecule PPI inhibitor identification remains challenging because of the lack of deep cavities at PPI interfaces. This article reports the discovery of a potent, cell-active small-molecule BCL6 inhibitor, BCL6-i (8), that operates through irreversible inhibition. First, we synthesized irreversible lead compound 4, which targets Cys53 in a cavity on the BCL6-BTB domain dimer by introducing an irreversible warhead to high-throughput screening hit compound 1. Further chemical optimization of 4 based on kinact/KI evaluation produced BCL6-i with a kinact/KI value of 1.9 × 104 M-1 s-1, corresponding to a 670-fold improvement in potency compared to that of 4. By exploiting the property of irreversible inhibition, engagement of BCL6-i to intracellular BCL6 was confirmed. BCL6-i showed intracellular PPI inhibitory activity between BCL6 and its corepressors, thus resulting in BCL6-dependent DLBCL cell growth inhibition. BCL6-i is a cell-active chemical probe with the most potent BCL6 inhibitory activity reported to date. The discovery process of BCL6-i illustrates the utility of irreversible inhibition for identifying potent chemical probes for intractable target proteins.

Discovery of artificial VIPR2-antagonist peptides possessing receptor- and ligand-selectivity.

Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41 nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47 nM (human), 180 nM (mouse), and 44 nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization.

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC50, KD, and thermodynamic parameters, the crystal structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery.

Novel DOCK2-selective inhibitory peptide that suppresses B-cell line migration.

Dedicator of cytokinesis 2 (DOCK2) is a key molecule for lymphocyte activation and migration. DOCK2 interacts with Ras-related C3 botulinus toxin substrate 1 (Rac1, GTPase) and mediates the GDP-GTP exchange reaction, indicating that inhibitors against protein-protein interaction (PPI) between DOCK2 and Rac1 would be good drug candidates for treating immune-related disorders. Here, we report DOCK2-selective PPI inhibitory peptides discovered using random peptide T7 phage display technology. These peptides inhibited DOCK2 activity at nanomolar concentrations and were delivered to intracellular compartments by combination with cell-penetrating peptide (CPP). Consequently, one peptide, R4-DCpep-2(V2W/K4R/ox)-NH2 (Ac-RRRRCWARYHGYPWCRRRR-NH2), inhibited migration in human B lymphocyte MINO cell line at IC50 = 120 nM. To our knowledge, this is the first report of a DOCK2-selective peptide inhibitor; this study will contribute to the development of novel DOCK2-targeting immunosuppressive drugs.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, KD and IC50 values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibited relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported.

Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis.

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

Amino-acid mutations of Gly12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 µM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

Discovery of peptidic miR-21 processing inhibitor by mirror image phage display: A novel method to generate RNA binding D-peptides.

A novel method to generate RNA binding D-peptide has been developed. To achieve the screening method, phage display was applied to "Mirrored" RNA (L-enantiomer of RNA). We have selected pre-miR21 as an initial screening target to demonstrate the method. The mirrored pre-miR-21 binding peptide sequences were successfully obtained, and were chemically synthesized using D-amino acids. D-peptide is expected to have favorable properties as a drug candidate such as protease resistance and low immunogenicity. As a result of binding evaluation of the D-peptide to pre-miR-21, the EC50 value was 440nM. In addition, the D-peptide possessed inhibition activity to miR-21 processing.

Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging.

A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu7, Ile9, and Asp12 are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.

Investigation on cellular uptake and pharmacodynamics of DOCK2-inhibitory peptides conjugated with cell-penetrating peptides.

Protein-protein interaction between dedicator of cytokinesis 2 (DOCK2) and Ras-related C3 botulinum toxin substrate 1 (Rac1) is an attractive intracellular target for transplant rejection and inflammatory diseases. Recently, DOCK2-selective inhibitory peptides have been discovered, and conjugation with oligoarginine cell-penetrating peptide (CPP) improved inhibitory activity in a cell migration assay. Although a number of CPPs have been reported, oligoarginine was only one example introduced to the inhibitory peptides. In this study, we aimed to confirm the feasibility of CPP-conjugation approach for DOCK2-inhibitory peptides, and select preferable sequences as CPP moiety. First, we evaluated cell permeability of thirteen known CPPs and partial sequences of influenza A viral protein PB1-F2 using an internalization assay system based on luciferin-luciferase reaction, and then selected four CPPs with efficient cellular uptake. Among four conjugates of these CPPs and a DOCK2-inhibitory peptide, the inhibitory activity of a novel CPP, PB1-F2 fragment 5 (PF5), conjugate was comparable to oligoarginine conjugate and higher than that of the non-conjugated peptide. Finally, internalization assay revealed that oligoarginine and PF5 increased the cellular uptake of inhibitory peptides to the same extent. Hence, we demonstrated that CPP-conjugation approach is applicable to the development of novel anti-inflammatory drugs based on DOCK2 inhibition by investigating both cellular uptake and bioactivity.

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects.

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

Discovery of a novel B-cell lymphoma 6 (BCL6)-corepressor interaction inhibitor by utilizing structure-based drug design.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes with corepressors via protein-protein interactions (PPIs). The complexes of BCL6 and corepressors play an important role in the formation of germinal centers (GCs), and differentiation and proliferation of lymphocytes. Therefore, BCL6-corepressor interaction inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting from high-throughput screening hits 1a and 2a, we identified a novel BCL6-corepressor interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC50=0.10µM, cell-based mammalian two-hybrid [M2H] assay IC50=0.72µM) by utilizing structure-based drug design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo studies.